Project description:We used a comprehensive quantitative proteomic profiling based on LC-MS/MS combined with TMT labeling strategy to analyze frozen-thawed Ashidan yak spermatozoa proteomic dynamic changes under three sequential conditions: density gradient centrifugation-based purification（FTH sperm）, incubation in capacitation medium(CAP sperm), and treatment with the calcium ionophore A23187 to induce the acrosome reaction process(AR sperm). FTH sperm Briefly, sperm-containing straws were thawed for 30 s at 37ºC prior to addition to a prepared 45-90% Percoll solution (Percoll P1644; 3.1mM KCl, 2.9mM NaH2PO4,80mM NaCl, 10mM HEPES, 2mM CaCl2·H2O, 0.4mM MgCl2·6H2O, 26mM Sodium DL-Lactate Solution, 25mM NaHCO3; pH 7.3-7.4) and diluted using modified Tyrode's Hepes buffered medium (Sp-TALPH, 114mM NaCl, 3.2mM KCl, 2mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 10mM HEPES, 3mg/ml BSA, 0.2mM Na pyruvate, 7.5x10-3mg/ml Gentamicin; pH 7.3-7.4) followed by centrifugation for 10 min at 700 xg to enable the isolation of highly motile sperm. To ensure that the gradient material was completely eliminated from these samples, the sperm cells collected from the bottom layer were washed two times using supplemented Sp-TALPH medium and centrifuged for 5 min at 300 xg. Sperm were then isolated and adjusted to a concentration of 100x106/mL using modified Tyrode's bicarbonate buffered medium (Sp-TALP; 114mM NaCl, 3.2mM KCl, 25mM NaHCO3, 0.4mM NaH2PO4·H2O, 10mM Na Lactate, 2mM CaCl2·H2O, 0.5mM MgCl2·6H2O, 6mg/ml BSA, 0.2mM Na pyruvate, 5x10-3mg/ml Gentamicin; pH 7.3-7.4). A CASA approach was then used to assess sperm motility, with only those samples exhibiting >70% total motility being retained for further analyses, as per previously published protocols. An aliquot containing 100 million FTH sperm was taken from each sample for subsequent protein isolation, with the remaining sperm being incubated in capacitation medium. CAP sperm Capacitation medium was composed of Sp-TALP containing heparin (50 ug/ml) and caffeine (5 mM), and sperm were incubated in this medium for 30 min at 38.5ºC in a 5%CO2 incubator, as these conditions have previously been shown to be optimal for the induction of capacitation in frozen-thawed yak sperm. An aliquot containing 100 million CAP sperm was taken from each sample for subsequent protein isolation. AR sperm AR induction was achieved by treating 990 uL capacitated sperm samples with 10 µL of A23187 (1.0mM in DMSO, CSNpharm, IL, USA) to a final concentration of 10 µM, followed by a 15 min incubation at 38.5ºC, For individual replicates, aliquots of 100 million AR sperm were collected for subsequent protein analyses.

Project description:Persistence of the invasive perennial weed leafy spurge is mainly attributed to seasonal production of underground adventitious buds (UABs), which undergo well-defined phases of dormancy (para-, endo- and eco-dormancy). These well-defined phases of dormancy also allow UABs of leafy spurge to survive extreme seasonal variations, including dehydration-stress. Consequently, objectives of this study include understanding the effects of dehydration-stress on vegetative reproduction, flowering competence, and transcript profiles at different phases of bud dormancy. The vegetative growth potential of UABs was monitored by removing the aerial portion of dehydration-stressed plants and re-watering the root system. Further, microarray analysis was used to follow transcriptome profiles to identify critical defense- and signaling-pathways at different phases of dormancy in UABs. Surprisingly, only 3 days of dehydration-stress is required to break the endodormant phase in UABs. In leafy spurge, vernalization of endodormant UABs has previously been shown to induce flower competence, while breaking of endodormancy via dehydration-stress did not induce floral induction. Thus, these two environmental treatments open a unique approach to independently dissect molecular mechanisms involved in endodormancy maintenance and floral competence. Bioinformatics analysis of transcriptome data helped to identify models and overlapping pathways. During endodormancy break, LEC1, PHOTOSYSTEM I RC, and brassinosteroids were identified as ooverlapping hubs of up-regulated genes, and DREB1A, CBF2, GPA1, MYC2, BHLH, BZIP, and flavonoids were identified as overlapping hubs of down-regulated genes. Additionally, key genes involved in metabolic activity, chromatin modification, and cross-talk between growth regulators were identified as playing a role during endodormancy maintenance. Plant material, controlled environmental treatments, and vegetative growth: Three-month-old paradormant leafy spurge plants grown in a greenhouse were acclimated for one week, under growth chamber conditions, prior to being subjected to a ramp-down in temperature (27 °C → 10 °C) and photoperiod (16 h → 8 h light) for 12 weeks to induce endodormancy, as previously established (Doğramacı et al. 2010; Foley et al. 2009). Endodormancy status was confirmed by comparing vegetative growth rates of plants with and without a ramp-down treatment. To study the effects of dehydration-stress on vegetative growth from UABs, water was withheld from endodormant plants up to 21 days. After dehydration for 0, 1-, 3-, 7-, 14-, and 21-days, the aerial portion of the plants were decapitated at the soil surface and vegetative growth and floral competence of UABs were monitored under growth-conducive conditions by re-watering the root system. Vegetative shoot growth from six plants was recorded weekly for four weeks, and results of four replications were analyzed using SAS 9.2 (SAS Institute, Cary, NC, 2008) software as described by Doğramacı et al. (2010). Microarray analysis: At the end of each treatment, crown buds were collected, and RNA extraction, cDNA synthesis, fluorescent labeling, microarray hybridization using ~23K element arrays, and spot intensity analyses were performed as previously described by Doğramacı et al. (2010). Based on unexpected vegetative growth results obtained from this study, only microarray data obtained from 0-, 1-, and 3-day dehydration-stressed endodormant UABs, which were compared with microarray data for endodormant, and flowering competent ecodormant UABs from our previous study (Doğramacı et al. 2010; GSE19217), were used for bioinformatics analyses. However, to merge the datasets from these two separate experiments, T-tests were first performed on microarray data obtained from endodormant UABs from each experiment, and differentially-expressed genes (p<0.005) were removed from the dataset (~5% of the spots). After removing the outliers, the two endodormant transcriptome data sets were grouped together and used as the baseline control for further ANOVA, T-tests and other analyses. Array normalization, statistical analyses and clustering of the dataset and Venn diagram generation were done using GeneMaths XT 5.1 software as previously described by Doğramacı et al. (2010).

Project description:Phosphorylation is a widespread post-translational modification that regulates numerous biological processes. Viruses can alter the physiological activities of host cells to promote virus particle replication, and manipulating phosphorylation is one of the mechanisms. Senecavirus A (SVA) the causative agent of porcine idiopathic vesicular disease. Although numerous studies on SVA have been performed, comprehensive phosphoproteomics analysis of SVA infection is lacking. In the present study, we performed a quantitative mass spectrometry-based phosphoproteomics survey of SVA infection in instituto biologico-rim suino-2 (IBRS-2) cells. Three parallel experiments were performed and 4520 phosphosites were identified on 2084 proteins. Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that many of the phosphorylated proteins were involved in apoptosis and spliceosome pathways, and subcellular structure localisation analysis revealed that more than half were located in the nucleus. Motif analysis of proteins with differentially regulated phosphosites showed that proline, aspartic acid and glutamic acid were the most abundant residues in the serine motif, while proline and arginine were the most abundant in the threonine motif. Forty phosphosites on 27 proteins were validated by parallel reaction monitoring phosphoproteomics, and 30 phosphosites in 21 proteins were verified. Nine proteins with significantly altered phosphosites were further explored, and eight (SRRM2, CDK13, DDX20, DDX21, BAD, ELAVL1, PBK, and STAT3) may play a role in SVA infection. Finally, identified eight predicted kinases, and the kinase activity of inhibitor of nuclear factor kappa-B kinase subunit β and CDK9 was reversed following SVA infection. This is the first phosphoproteomics analysis of SVA infection of IBRS-2 cells, and the results greatly expand our knowledge of SVA infection. The findings provide a basis for studying the interactions of other picornaviruses and their mammalian host cells.

			Action	DRS
	0143_.msf	Msf
	0143_.msfView	Msf
	0143_.pdResult	Other
	0143_.pdResultView	Other
	0143_.pdStudy	Other

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Evolutionarily conserved E3 ubiquitin ligase modulates carotenoid, sterol and lipid crosstalk

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